Process for isolation of osteopontin from milk

ABSTRACT

A process for isolation of milk osteopontin from a material containing milk osteopontin by optionally mixing the milk material with a calcium source and separate the osteopontin containing phase from the rest of the milk material by pH adjustment.

The present invention relates to a new process for isolation ofosteopontin from milk.

Osteopontin (OPN) is a secreted adhesive glycophosphoprotein originallyisolated from the collagenous extra cellular matrix of mineralized bone(Franzén A, Heineg{dot over (a)}rd, D. 1985. Isolation andcharacterization of two sialoproteins present only in bone calcifiedmatrix. Biochem. J. 232:715-724.). In the recent years, osteopontin hasbeen found to be expressed by a number of different cell types includingosteoblasts, arterial smooth muscle cells, leukocytes, several types ofepithelial cells and transformed cells of different lineages (Denhardt DT, Butler W T, Chambers A F, Senger D R. (eds.). 1995. Osteopontin: rolein cell signalling and adhesion. Ann. N.Y. Acad. Sci., 760).Accordingly, OPN has been detected in many tissues including kidney,placenta, secretory epithelia and ganglia of the inner ear, and smoothmuscle of the vascular system (Butler W T, Ridall A L, McKee M D. 1996.Osteopontin. In Bilezekian J P, Rai L G, Rodan G A (eds.) Principles ofbone biology. Academic Press, San Diego, Calif., U.S.A., pp. 167-181.Furthermore OPN is also present in many body fluids, notably plasma,urine, bile and milk, and it displays elevated expression in manytransformed cells (Senger D R, Peruzzi C A, Gracey C F, Papadopoulos A,Tenen D G. 1988. Secreted phosphoproteins associated with neoplastictransformation: close homology with plasma proteins cleaved during bloodcoagulation. Cancer Res. 48: 5770-5774). Osteopontin is highly acidicwith approximately 25% of the amino acid being aspartate/aspartic acidand glutamate/glutamic acid as well as a significant number ofphosphorylated amino acids (Sørensen E S, Petersen T E. 1994Identification of two phosphorylation motifs in bovine osteopontin.Biochem. Biophys. Res. Commun. 198:200-205; Sørensen, E S, Højrup, P,Petersen, T E. 1995. Posttranslational modifications of bovineosteopontin: Identification of twenty-eight phosphorylation and threeO-glycosylation sites. Protein Sci. 4:2040-2049).

Osteopontin contains an RGD (arginine, glycine, aspartate)integrin-binding sequence, and it can promote attachment of cells tovarious surfaces, for example the attachment of osteoblasts to boneduring bone remodelling. In addition to cell attachment capability,osteopontin can act as a cytokine. Other proposed uses or roles forosteopontin include chemotaxis and inhibition of nitric oxide synthaseexpression.

Thus, osteopontin has been proposed for use as a pharmaceutical agent.EP 705842 proposes the use of osteopontin in diagnosis, prophylaxis andtherapy of osteoarthritis and rheumatiod arthritis. Osteopontin is alsobelieved to play a role in enhancing bone growth and wound healing inmammals, cf. for example EP 942452 and WO 9933415. Further, osteopontinhas been proposed for inhibition of nitric oxide, cf. U.S. Pat. No.5,695,761. Osteopontin has also been proposed for solubilization ofdivalent metal ions for addition to foods, cf. abstract of JP 9173018.

Osteopontin has been isolated in research scale amounts (micrograms tolow milligram's scale) from a number of tissues and fluids, includingmineralized bone. However, there is a huge demand for osteopontin forexperimental as well as for industrial use. All known processes forisolating osteopontin are at experimental scale giving too small amountsfor industrial use.

Sørensen, E S, Petersen T. 1993. Journal of Dairy Research 60, 189-197,Purification and characterization of three proteins isolated from theproteose peptone fraction of bovine milk describes a method involvingTCA, trichloro acetic acid, precipitation of proteins, This method isnot compatible with production of food ingredients, because TCA is notallowed in food products. Furthermore, the method includes gelfiltration, which is not suitable for large-scale production.

Bayless K J, Davis G E, Meininger G A. 1997. Protein Expression andPurification 9, 309-314, Isolation and biological properties ofosteopontin from bovine milk describes a method including ion-exchangeand two steps of hydrophobic chromatography on phenyl-separose columns.The complexity of the process, and especially the hydrophobicchromatography makes the method inapplicable for large-scalepurification of osteopontin.

Senger D R, Peruzzi C A, Papadopoulos A, Tenen D G. 1989. Biochimica etBiophysica Acta 996, 43-48. Purification of a human milk protein closelysimilar to tumor-secreted phosphoproteins and osteopontin proposes apurification method. The purification method described for human milkinvolves barium citrate affinity as well as reverse phase HPLCchromatography, which makes it inapplicable for large-scalepurification.

The present invention solves the above problems. The invention disclosesa process for large-scale purification or isolation of osteopontin frombovine milk (and milk from other domesticated milk producing mammalse.g. goat, sheep, buffalo, lama, camel, etc.). Milk osteopontin ishighly preferred because it occurs naturally in milk from domesticanimals. According to the invention osteopontin is isolated bytechniques approved for dairy foods production. This food-gradeosteopontin can therefore be used as an ingredient in food products forhuman consumption without any risks.

The present invention proposes a process for isolation of osteopontinfrom milk, wherein a milk material containing osteopontin and a materialcontaining calcium are mixed and pH is adjusted to keep osteopontin insolution while precipitating other milk constituents or to aggregate orbind osteopontin while removing other soluble constituents.

The process can be performed in one or more steps. It is also possibleto combine the steps of keeping osteopontin in solution and binding oraggregating it.

In the process of the invention the raw material is preferably based onmilk containing osteopontin. Whey is a good raw material, since thecasein proteins contained therein have been removed. Especially wheyoriginating from chemical acidifying of milk is suitable, because itsosteopontin is intact. On the other hand, the osteopontin contained incheese whey can be partially hydrolysed. However, it is not yet knownwhether this reduces or completely destroys the properties ofosteopontin.

Separation of osteopontin from denatured and precipitated protein can beperformed by microfiltration or centrifugation as normally performed inthe dairy industry.

The pore size can be from 0.1 to 1.4 μm. Ceramic filters are especiallywell suited owing to mechanical stability and long life. The separationcan take place at temperatures from 10 to 80° C. 50-55° C. areespecially well suited owing to great capacity and stablebacteriological conditions.

All types of anion exchangers can be used. Here a DEAE Sepharose fastflow exchanger is used. During the ion exchange pH can vary from approx.3 to approx. 6.

The steps involving (A) solution or solubilization of osteopontin or (B)aggregation or binding of osteopontin can be combined in one process.The product streams can be treated in different ways before and/or afterthese steps to concentrate, separate, dry or perform other processescommonly used in the diary industry. Thus e.g. microfiltration can beused. The person skilled in the diary art will easily determine a properfilter, cf. e.g. Tetra Paks “Dairy processing Handbook” (1995), pp123-132.

Normally it is preferred to concentrate whey before starting the processof the invention to reduce the amount of water to treat. Forpreconcentration of whey any conventional ultrafiltration system can beused: plate and frame; hollow fibre, tubular, ceramic and spiral, etc.Spiral systems are especially well suited from an economic point of viewfor the time being. Any membrane which does not allow osteopontin topass through the membrane is suitable. The pore size of such membranesis 20,000 D or less. Suitable membranes are e.g. Koch HFK131, Desal PWor similar membranes.

The soluble Ca source can be any soluble calcium compound, such ascalcium hydroxide, calcium chloride or calcium acetate. Calcium nitriteand calcium nitrate is also useable, but will normally not berecommended, if osteopontin is to be used in the food industry.

To aggregate or bind osteopontin to an insoluble Ca source use can bemade of Ca₃PO₄ or other insoluble Ca source. pH adjustment toprecipitate calcium phosphate or another insoluble salt with osteopontincan be within the range 6.0-8.5. The pH range 6.5-8.0 is particularlywell suited. Especially, pH about 7.0 is suitable. Any base can be used;organic as well as inorganic, as pH adjustment agent in this process.Especially the bases NaOH, KOH, Ca(OH)₂ are suitable. Especially NaOH issuitable.

pH adjustment to keep osteopontin in solution is 3.5 to 5.0, preferably4.0-4.6. pH 4.2 is most preferred. For the pH adjustment any organic orinorganic acid can be used. Hydrochloric acid is especially suitableowing to strength and price. If the milk starting material containingosteopontin also contains natural calcium from the milk less calcium isto be added or no calcium at all. Also, use of Ca(OH)₂ for pH adjustmentcan minimise the amount of other Ca source. Normally a suitable amountwill be one where the concentration of calcium in the solution is 0.2%.There is no lower limit, but less than about 0.05% per protein % willgive a reduced yield of osteopontin. 0.1% of calcium will be effective,but the yield of osteopontin is reduced compared to using 0.2% ofcalcium. Concentrations of calcium up to 0.4% have been tried. However,only little increase is obtained by concentrations above 0.2%.Therefore, 0.2% is preferred, but also 0.3% could be used.

Separation of precipitated calcium phosphate or another solid Ca sourcecontaining osteopontin can be separated from the rest by any usualmethod, such as microfiltration or centrifugation. The pore size can befrom 0.1 to 1.4 μm. Ceramic filters are especially well suited owing tomechanical stability and long life. The separation can take place attemperatures from 10 to 80° C. 50-55° C. are especially well suitedowing to great capacity and stable bacteriological conditions.

For the separation of osteopontin from dissolved calcium salt anyultrafiltration system can be used: plate and frame; hollow fibre,tubular, ceramic and spiral, etc. Spiral systems are especially wellsuited from an economic point of view. Any membrane which does not allowosteopontin to pass through the membrane can be used. The nominal poresize of such membranes is typically 20,000 D or less. Suitable membranesare e.g. Koch HFK328, Desal PV or similar membranes. All types of anionexchangers can be used. Here DEAE Sepharose fast flow exchanger is used.

During the ion exchange pH can vary from approx. 3 to approx. 6.

Germ filtration of whey protein concentrate, WPC, retentate before thepH adjustment to 7.0 in order to precipitate Ca₃(PO₄)₂ or another solidwith oseopontin produces a purer aggregate, which will also give a purerproduct for further processing in the processes and thus also a littlepurer osteopontin products.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE 1

1000 kg of casein whey with pH 4.55, 0.53% protein, approximately 20 ppmosteopontin (0.002%), and 4.50% dry matter are concentrated in a spiralultrafiltration plant with membranes having a nominal pore size so thatthe proteins do not pass through the membrane. Typical nominal pore sizeis 10,000 D. The temperature during the filtration is 50° C. and themean pressure is 3.5 bars. Concentration is carried on until 900 kg ofpermeate, which do not contain osteopontin, are removed. During theconcentration mean flux is 45.6 litres/m²/h. 100 kg of retentate with pH4.55, 3.86% protein, approx. 200 ppm osteopontin (0.02%), and 10.3% drymatter are left.

The 100 kg of retentate are pasteurized at 68° C. for 15 sec and cooledto 50° C. Hereafter pH is adjusted to 7.0 with 6% NaOH.

After being allowed to stand for 2 hours, microfiltration is performedon 0.1.4 μm ceramic membranes at 50° C. with a mean pressure of 4.0bars. Diafiltration is carried out with 50° C. hot demineralized waterpH adjusted to 7.0 until the conductivity in the permeate is below 100μS. During the entire filtration the mean flux is 310 litres/m²/h.

20,0 kg of retentate from the microfiltration are collected and cooledto 8° C. in ice water, with pH 7.0, 0.58% protein, approx. 1000 ppmosteopontin (0.1%), and 4.1% dry matter. The retentate is spray dried ina NIRO tower drier and 0.7 kg of powder with 13.6% protein, approx. 2.3%osteopontin, and 96.4% dry matter is obtained. The dry matter consistsof 80% ashes, 27% calcium and 13.8% phosphorus.

EXAMPLE 2

2000 kg of casein whey with pH 4.55, 0.55% protein, approximately 20 ppmosteopontin (0.002%), and 4.53% dry matter are concentrated in a spiralUF plant with membranes having a nominal pore size so that the proteinsdo not pass through the membrane. Typical pore size is 10,000D. Thetemperature during the filtration is 12° C. and the mean pressure is 3.5bars. Concentration is carried on until 1800 kg of permeate, which donot contain osteopontin, are removed. During the concentration mean fluxis 27.6 litres/m²/h. 200 kg of retentate with pH 4.55, 3.97% protein,approx. 200 ppm osteopontin (0.02%), and 10.4% dry matter are left.

The 200 kg of retentate are pasteurized at 68° C. for 15 sec and cooledto 50° C. Hereafter pH is adjusted to 7.0 with 6% NaOH.

After being allowed to stand for 30 min, microfiltration is carried outon 0.2 m² 1.4 μm ceramic membranes at 50° C. with a mean pressure of 4.0bars. Diafiltration is performed with 50° C. hot demineralized water pHadjusted to 7.0 until the conductivity in the permeate is below 100 μS.During the entire filtration the mean flux is 310 litres/m²/h. 20.0 kgof retentate from the microfiltration are collected and cooled to 8° C.in ice water, with pH 7.0, 1.25% protein, approx. 2,000 ppm osteopontin(0.2%), and 8.3% dry matter.

The retentate is pH adjusted to 3.0 with 28% hydrochloric acid, whichproduces an almost clear solution.

This pH 3.0 adjusted solution is ultrafiltered at 10° C. and the meanpressure 4.0 bars on a membrane with a cut-off value of 5,000D. Theretentate is diafiltered with demineralized water until the conductivityin the permeate is below 0.1 μS. During the entire filtration the meanflux is 17.8 litres/m²/h. 10 kg of retentate with 2.42% protein, approx.4,000 ppm osteopontin (0.4%), and 2.6% dry matter are collected.

The retentate is spray dried in a NIRO tower drier and 0.2 kg of powderwith 89.6% protein, approx. 14.8% osteopontin, and 96.4% dry matter isobtained.

EXAMPLE 3

1000 kg of preconcentrated casein whey with pH 4.55, 3.76% protein,approx. 200 ppm osteopontin (0.02%), and 10.3% dry matter arepasteurised at 67° C. for 15 sec and cooled to 50° C. Hereafter pH isadjusted to 7.0 with 6% NaOH.

After being allowed to stand for 60 min, microfiltration is carried outon 2.8 m² 1.4 μm ceramic membranes at 50° C. with a mean pressure of 4.0bars. Diafiltration is performed with 50° C. hot demineralized water pHadjusted to 7.0 until the conductivity in the permeate is below 100 μS.During the whole filtration the mean flux is 325 litres/m²/h.

100 kg of retentate from the microfiltration with pH 7.0, 1.22% protein,approx. 2,000 ppm osteopontin (0.2%), and 8.3% dry matter are collectedand cooled to 5° C. on a plate heat exchanger.

The retentate is pH adjusted to 3.0 with 28% hydrochloric acid, whichproduces an almost clear solution. This pH 3.0 adjusted solution isultrafiltered at 10° C. and the mean pressure 4.0 bars on a membranewith a cut-off value of 5,000D. The retentate is diafiltered withdemineralized water until the conductivity in the permeate is below 100μS. Then diafiltration is performed with 0.45 M KH₂PO₄ with pH=4.5 untilpH is 4.5 in the retentate. During the entire filtration the mean fluxis 15.9 litres/m²/h. 10 kg retentate with 12.1% protein, approx. 20,000ppm osteopontin (2.0%), and 13.4% dry matter are collected.

The retentate is pumped through a column with an anion exchanger whichis equilibrated with 0.45 M KH₂PO₄ with pH=4.5. Thus osteopontin isbound to the column while the greater part of other whey proteins is notbound. The column is washed with 0.45 M KH₂PO₄ with pH=4.5 until theabsorption at 280 nm is 0.

Osteopontin is eluted from the anion exchanger with 0.7 M KH₂PO₄ withpH=4.5 until the absorption at 280 nm is 0 and 50 litres of eluatecontaining 500 g protein, of which 40% is osteopontin, are collected.The eluate is concentrated and diafiltered to remove salts onultrafiltration membranes having a pore size of 5,000D, at 10° C. andthe mean pressure 4.0 bars. 5 kg of retentate with 10.7% dry matter,approx. 4% osteopontin, and 9.6% protein are collected. The retentate isspray dried in a NIRO tower drier and 0.5 kg of powder with 86.0%protein, approx. 36% osteopontin, and 95.8% dry matter is obtained.

EXAMPLE 4

1000 kg of casein whey with pH 4.56, 0.52% protein, approx. 20 ppmosteopontin (0.002%), and 4.50% dry matter are pasteurised at 71° C. for15 s and cooled to 50° C. Hereafter pH is adjusted to 7.0 with 6% NaOH.

After being allowed to stand for 2 hours, microfiltration is performedon 1.4 m² 1.4 μm ceramic membranes at 50° C. with a mean pressure of 4.0bars. Diafiltration is performed with 50° C. hot demineralized water pHadjusted to 7.0 until the conductivity in the permeate is below 100 μS.During the entire filtration the mean flux is 520 litres/m²/h.

30.0 kg of retentate from the microfiltration are collected and cooledto 8° C. with ice water, with pH 7.0, 0.78% protein, approx. 670 ppmosteopontin (0.07%), and 16.1% dry matter. The retentate is spray driedin a NIRO tower drier and 4.5 kg of powder with 4.7% protein, approx.0.4% osteopontin, and 96.4% dry matter are obtained. The dry matterconsists of 85% ashes, 27% calcium and 13.8% phosphorus.

EXAMPLE 5

5000 kg of casein whey with pH 4.56, 0.51% protein, approx. 20 ppmosteopontin (0.002%), and 4.50% dry matter are pasteurised at 69° C. for15 sec and cooled to 50° C. Hereafter pH is adjusted to 7.0 with 6%NaOH.

After being allowed to stand for 2 hours, microfiltration is performedon 1.4 m² 1.4 μm ceramic membranes at 50° C. with a mean pressure of 4.0bars. Diafiltration is carried out with 50° C. hot demineralized waterpH adjusted to 7.0 until the conductivity in the permeate is below 100μS. During the entire filtration the mean flux is 550 litres/m²/h.

150 kg of retentate from the microfiltration are collected and cooled to8° C. in ice water, with pH 7.0, 0.73% protein, approx. 670 ppmosteopontin (0.07%), and 16.4% dry matter.

The retentate is pH adjusted to 3.0 with 28% hydrochloric acid, whichproduces an almost clear solution. This pH 3.0 adjusted solution isultrafiltered at 10° C. and the mean pressure 4.0 bars on a membranewith a cut-off value of 10,000D. The retentate is diafiltered withdemineralized water until the conductivity in the permeate is below 100μS. During the entire filtration the mean flux is 25.3 litres/m²/h. 10kg of retentate with 11.0% protein, approx. 10,000 ppm osteopontin(1.0%), and 12.6% dry matter are collected.

The retentate is spray dried in a NIRO tower drier and 1.1 kg of powderwith 83.8% protein, approx. 7.6% osteopontin, and 96.2% dry matter areobtained.

EXAMPLE 6

10,000 kg of casein whey with pH 4.56, 0.54% protein, approx. 20 ppmosteopontin (0.002%), and 4.50% dry matter are pasteurised at 69° C. for15 sec and cooled to 50° C. Hereafter pH is adjusted to 7.0 with 6%NaOH.

After being allowed to stand for 2 hours, microfiltration is performedon 2.8 m² 1.4 μm ceramic membranes at 50° C. with a mean pressure of 4.0bars. Diafiltration is carried out with 50° C. hot demineralized waterpH adjusted to 7.0 until the conductivity in the permeate is below 100μS. During the entire filtration the mean flux is 570 litres/m²/h.

300 kg of retentate from the microfiltration are collected and cooled to8° C. in ice water, with pH 7.0, 0.76% protein, approx. 670 ppmosteopontin (0.07%), and 16.3 dry matter.

The retentate is pH adjusted to 3.0 with 28% hydrochloric acid, whichproduces an almost clear solution. This pH 3.0 adjusted solution isultrafiltered at 10° C. and the mean pressure 4.0 bars on a membranewith a cut-off value of 10,000D. The retentate is diafiltered withdemineralized water until the conductivity in the permeate is below 100μS. During the entire filtration the mean flux is 24.6 litres/m²/h. 20kg of retentate with 10.8% protein, approx. 10,000 ppm osteopontin(1.0%), and 12.3% dry matter are collected.

The retentate is pumped through a column with an anion exchanger whichis equilibrated with 0.45 M KH₂PO₄ with pH=4.5. The osteopontin is thusbound to the column, while the greater part of other whey proteins isnot bound. The column is washed with 0.45 M KH₂PO₄ with pH=4.5 until theabsorption at 280 nm is 0.

Osteopontin is eluted from the anion exchanger with 0.7 M KH₂PO₄ withpH=4.5 until the absorption at 280 nm is 0 and 50 litres of eluatecontaining 560 g of protein, of which 36% is osteopontin, are collected.The eluate is concentrated and diafiltered to remove salts onultrafiltration membranes having a pore size of 5,000D, at 10° C. andthe mean pressure 4.0 bars. 5 kg of retentate with 12.3% dry matter,approx. 4% osteopontin, and 11.2% protein are collected. The rententateis spray dried in a NIRO tower drier and 0.6 kg of powder with 87.6%protein, approx. 31% osteopontin, and 96.2% dry matter is obtained.

EXAMPLE 7

10,000 kg of casein whey with pH 4.53, 0.55% protein, approx. 20 ppmosteopontin (0.002%), and 4.50% dry matter are ultra-/diafiltered in aspiral UF plant with membranes having a nominal pore size so that theproteins do not pass through the membrane. Typical pore size is 20,000D.The temperature during the filtration is 15° C. and the mean pressure is3.5 bars. Once the filtration is terminated, 152 kg of retentate with pH4.54, 23.1% protein, approx. 1,300 ppm osteopontin (0.13%), and 28.7%dry matter are collected. During the concentration mean flux is 25.6litres/m²/h. The 152 kg of retentate are diluted with 722 kg ofdemineralized water so that the protein content is 4.0%. pH is adjustedto 7.4 with 6% NaOH. Then heat treatment is performed at 85° C. for 30minutes and cooling to 8° C. is carried out. 6.4 kg of CaCl_(2,)2H₂O isadded to the heat treated retentate, and pH is adjusted to 4.2 with 6%hydrochloric acid.

After being allowed to stand for at least 2 hours (or to the next day),heating to 50° C. is performed and microfiltration is carried out on 1.4m² 0.8 μm ceramic membranes at 50° C. with a mean pressure of 4.0 bars.Diafiltration is carried out with 50° C. hot demineralized water pHadjusted to 4.2 until the conductivity in the permeate is below 100 μS.During the filtration the mean flux is 415 litres/m²/h. A total of 2,000litres of permeate having continuously being cooled to 8° C. over aplate heat exchanger (PVV), with pH 4.2, 0.16% protein, approx. 100 ppmosteopontin (0.01%), and 4.1% dry matter is collected.

The permeate from the microfiltation is pH adjusted to 6.6 with 6% NaOHand it is ultra/diafiltered in a spiral UF plant at 10° C. with a meanpressure of 4.0 bars until the conductivity in the permeate is below 500μS. A filtration on membranes having a pore size of 5,000D is carriedout. 40 kg of retentate with pH 6.5, 7.9% protein, approx. 5,000 ppmosteopontin (0.5%), and 9.6% dry matter is collected.

The retentate is spray dried in a NIRO tower drier and 3.5 kg of powderwith 79.2% protein, approx. 5.0% osteopontin, and 96.2% dry matter isobtained.

EXAMPLE 8

200 kg of casein whey retentate with pH 4.55, 23.2% protein, approx.1,300 ppm osteopontin (0.13%), and 29.1% dry matter are diluted with1,000 kg of demineralized water and pH is adjusted to 7.4 with 6% NaOH.Then heat treatment is carried out at 85° C. for 30 min and cooling to8° C. is performed. 8.8 kg of CaCl₂, 2H₂O are added to the heat treatedretentate; and pH is adjusted to 4.1 with 6% hydrochloric acid.

After standing for at least 2 hours (or to the next day), heating to 50°C. is performed and microfiltration is carried out on 2.8 m² 0.8 μmceramic membranes at 50° C. with a mean pressure of 4.0 bars.Diafiltration is performed with 50° C. hot demineralized water pHadjusted to 4.1 until the conductivity in the permeate is below 100 μS.During the entire filtration the mean flux is 445 iitres/m²/h. A totalof 2,800 litres of permeate having been cooled to 8° C. over a plateheat exchanger (PVV), with pH 4.1, 0.16% protein, approx. 100 ppmosteopontin (0.01%), and 4.0% dry matter is collected.

The permeate from the microfiltration is pH adjusted to 6.6 with 6% NaOHand it is ultra/diafiltered in a spiral UF plant at 10° C. with a meanpressure of 4.0 bars until the conductivity in the permeate is below 500μS. A filtration is carried out on membranes having a pore size of5,000D. 40 kg of retentate with pH 6.5, 11.2% protein, approx. 6,500 ppmosteopontin (0.65%), and 12.4% dry matter is collected.

The retentate is pumped through a column with an anion exchanger whichis equilibrated with 0.45 M KH₂PO₄ with pH=4.5. Osteopontin is thusbound to the column, while the greater part of other proteins is notbound. The column is washed with 0.45 M KH₂PO₄ with pH=4.5 until theabsorption at 280 nm is 0.

Osteopontin is eluted from the anion exchanger with 0.7 M KH₂PO₄ withpH=4.5 until the absorption at 280 nm is 0 and 50 l of eluate containing500 g of protein, of which 50% is osteopontin, are collected. The eluateis concentrated and diafiltered to remove salts on ultrafiltrationmembranes having a pore size of 5,000D, at 10° C. and the mean pressure4.0 bars. 5 kg of retentate with 10.7% dry matter, approx. 5%osteopontin, and 9.6% protein are collected. The retentate is spraydried in a NIRO tower drier and 0.5 kg of powder with 86.0% protein,approx. 45% osteopontin, and 95.8% dry matter is obtained.

1. A process for purification of osteopontin from milk comprising mixinga milk material containing osteopontin and a material containing calciumand adjusting pH to keep osteopontin in solution while precipitatingother milk constituents or adjusting pH to aggregate or bind osteopontinwhile removing other milk constituents in solution.
 2. A processaccording to claim 1, wherein the milk material is mixed with a solublecalcium salt and pH is adjusted to an acid pH to keep the osteopontin insolution while precipitating other milk constituents.
 3. A processaccording to claim 2, wherein pH is adjusted to pH 3.5 to 5.0.
 4. Aprocess according to claim 2, wherein pH is adjusted to pH 4.0 to 4.6.5. A process according to claim 2, wherein pH is adjusted to pH about4.2.
 6. A process according to claim 2, wherein the soluble calciumsource is calcium chloride.
 7. A process according to claim 1, whereinthe milk material is mixed with an insoluble calcium salt and pH isadjusted to a neutral to basic pH to bind or aggregate the osteopontinwhile precipitating it without precipitating the major part to the othermilk constituents.
 8. A process according to claim 7, wherein pH isadjusted to pH 6.0 to 8.5.
 9. A process according to claim 7, wherein pHis adjusted to pH 6.5 to 8.0.
 10. A process according to claim 7,wherein pH is adjusted to pH about
 7. 11. A process according to claim1, wherein the mixing and adjusting steps are carried out sequentially.12. A process according to claim 7, wherein, in separate steps, a milkmaterial containing osteopontin is mixed with an insoluble calcium saltand the pH is adjusted to a neutral or basic pH to bind or aggregate theosteopontin while precipitating it without precipitating the major partof the other milk constituents.
 13. A process according to claim 1,wherein the milk material containing osteopontin is a whey product. 14.A process according to claim 1, wherein the milk material containingosteopontin is a concentrated whey product.
 15. A process according toclaim 1, wherein the milk material containing osteopontin is a wheyproduct from chemical acidification of milk.
 16. A process according toclaim 2 or 7, wherein the milk material containing osteopontin and thematerial containing calcium are mixed so that the concentration ofcalcium in the mixture is about 0.05-0.3% based on protein weight %. 17.A process according to claim 16, wherein the milk material containingosteopontin and the material containing calcium are mixed so that theconcentration of calcium in the mixture is about 0.2% based on proteinweight %.
 18. A process for purification of osteopontin from milkcomprising adjusting the pH of a milk material containing osteopontinand calcium to keep osteopontin in solution while precipitating othermilk constituents or to aggregate or bind osteopontin while removingother milk constituents in solution.
 19. The process according to claim18, wherein the pH is adjusted to pH 4.0 to 4.6 to keep the osteopontinin solution while precipitating other milk constituents.
 20. The processaccording to claim 18, wherein the pH is adjusted to pH 6.5 to 8.0 tobind or aggregate the osteopontin while precipitating it withoutprecipitating the major part of the other milk constituents.